Messenger RNA localization plays a key role in creating the asymmetric distributions of proteins necessary for cellular and developmental polarity. The sorting of specific mRNAs to different subcellular domains is a complex process involving the assembly and trafficking of large ribonucleoprotein (RNP) complexes. How specificity is conferred on this process, particularly in cells where several mRNAs localization pathways operate concurrently, is poorly understood. This proposal integrates biochemical, genetic, and imaging-based approaches to investigate how mRNAs are specifically recognized and packaged into localization competent particles, how these RNP particles are adapted to different localization mechanisms, the extent to which localization pathways are interconnected, and how multiple localization pathways are coordinated within a single cell. The Drosophila oocyte and early embryo provide ideal model systems for these studies because they are equipped with several mechanistically distinct trafficking pathways that direct the localization of mRNAs essential for axis formation and germline development. In Aim 1, we will investigate mechanisms of RNP particle assembly using biochemical approaches including tandem RNA affinity purification to isolate and characterize the components of Nanos RNP complexes. These studies will be complemented in Aim 2 by two imaging-based approaches that investigate whether concurrent trafficking pathways are coordinated through the sharing of RNP particles by different mRNAs. Aim 3 expands our studies to determine the broader significance of mRNA localization in the development and function of polarized cells through a novel genome- wide screen for transcripts with asymmetric subcellular distributions in neurons. The identification of new localized mRNAs in this screen and screens in other cell types performed by our collaborators will shed light on determinants of mRNA targeting specificity and may uncover novel roles for localized mRNAs in development and function of polarized cells.